This proposal focuses on the mechanism underlying non-invasive cellular import of peptides utilizing the hydrophobic region of the signal sequence as a targeted cell membrane translocating motif. Such import represents an increasingly utilized approach to study intracellular protein-protein and protein-DNA interactions involved in signal transduction, intracellular trafficking of proteins, and gene transcription. This technique obviates the need for pore-forming membrane permeabilizing reagents or microinjection of individual cells. The analysis will be accomplished using intact cultured erythroleukemia, endothelial, and intestinal epithelial cells and phospholipid vesicles. The hypothesis that peptide import involves translocation through the phospholipid bilayer without involvement of specific chiral receptor or transporter will be examined. The role of membrane cholesterol in cell permeable peptide import will be analyzed. Its kinetics will be determined in regard to peptide chirality and amphiphilic and helical determinants of synthetic peptides. Improved synthesis of functional peptides based on the convergent scheme of the domain ligation strategy will allow facile examination of multiple peptide analogs of membrane- translocating sequence in systematic structure-function analysis. This analysis will include the determination of the minimal length membrane permeable sequence and all D-, retro-, and retro-inverso analogs of the model membrane translocating sequence based on the hydrophobic region of the signal sequence. The next level of analysis will focus on the turnover (half-time) of imported peptides, the role of extracellular and intracellular degradation, and redistribution with a special emphasis on the role of blood cells and transendothelial and transepithelial transport. Finally, analysis of cellular import will focus on targeting functional peptides and peptide-oligonucleotide complexes to subcellular compartments such as nuclei and mitochondria to regulate gene expression and programmed cell death. Thus, implicit to this fundamental mechanism of cellular import of functional peptides and peptide-oligonucleotide complexes will be improved non-invasive delivery of bioactive molecules into living cells.